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Single cell–resolution western blotting

Nature Protocols volume 11, pages 1508–1530 (2016)Cite this article

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Abstract

This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. Like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. Once the microdevice has been fabricated, the assay can be completed in 4–6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. The technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine.

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Figure 1: Single-cell western blotting (scWB) workflow and principles.
Figure 2: Real-time imaging of in-well chemical lysis of GFP-expressing U373 glioblastoma cells (U373-GFP).
Figure 3: Selection of suitable PAGE separation conditions.
Figure 4: One-step grayscale photopatterning of a scWB device creates 1-mm-long pore-gradient microgels, with each large-to-small-pore-size gel aligned to a microwell.
Figure 5: Optimization of PAGE separation performance depends on the scWB device geometry and electrical interfacing.
Figure 6: scWB PAG slide fabrication.
Figure 7: Pore-gradient PAG slide fabrication.
Figure 8: Examples of poor and ideal single-cell settling into microwells.
Figure 9: Handling of the scWB device during immunoprobing.
Figure 10: The scWB image analysis workflow.
Figure 11: scWB reports GAPDH and βTub expression in single U373 glioblastoma cells.
Figure 12: Comparison of uniform versus pore-gradient scWB readouts.

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Acknowledgements

Research reported in this publication was supported in part by the National Cancer Institute of the National Institutes of Health (R21CA183679 to A.E.H.), a New Innovator Award from the National Institutes of Health (1DP2OD007294 to A.E.H.), an NSF CAREER award from the National Science Foundation (CBET-1056035 to A.E.H.), a Diversity Supplement from the National Institutes of Health (to E.S.) and National Science Foundation Graduate Research Fellowships (DGE 1106400 to K.A.Y., J.V. and T.A.D.). We are grateful to S. Kumar's laboratory in the Department of Bioengineering, University of California, Berkeley, for providing the U373 MG and U373-GFP cell lines. We acknowledge the helpful feedback from students enrolled in the 2015 Single Cell Analysis course at Cold Spring Harbor Laboratory.

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Author notes

  1. Kevin A Yamauchi and Julea Vlassakis: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Bioengineering, University of California, Berkeley, California, USA

    Chi-Chih Kang, Kevin A Yamauchi, Julea Vlassakis, Elly Sinkala, Todd A Duncombe & Amy E Herr

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  1. Chi-Chih Kang
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Contributions

All authors designed the experiments. C.-C.K., K.A.Y., J.V. and E.S. performed the experiments. C.-C.K. and T.A.D. performed the data analysis. All authors wrote the manuscript.

Corresponding author

Correspondence to Amy E Herr.

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Competing interests

All co-authors are co-inventors on intellectual property related to the device and assay described here and may benefit from royalties from licensing.

Integrated supplementary information

Supplementary Figure 1 Dimensioned scWB silicon wafer mold drawing.

Example wafer mold design for the scWB. The wafers are fabricated using standard photolithography techniques with SU-8 2025 photoresist. As SU-8 2025 is a negative photoresist, regions of the mask containing features to be polymerized (e.g. the micropillars) should be transparent. The photomasks are printed on mylar.

Supplementary Figure 2 Dimensioned drawing of the scWB electrophoresis chamber.

The scWB electrophoresis chamber is fabricated out of Acrylonitrile butadiene styrene with a fused deposition molding 3D printer (e.g. MakerBot Replicator 2x). The chamber was printed with the cavity facing up and with a raft composed of dissolvable filament. The solid models was produced in SolidWorks and preprocessed in MakerWare.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2, Supplementary Note and Supplementary Table 1 (PDF 523 kb)

Supplementary Data 1

30-μm Microwells (ZIP 28 kb)

Supplementary Data 2

EPchamber_v01 (ZIP 5 kb)

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Kang, CC., Yamauchi, K., Vlassakis, J. et al. Single cell–resolution western blotting. Nat Protoc 11, 1508–1530 (2016). https://doi.org/10.1038/nprot.2016.089

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